Adenoviruses are among the most commonly used vectors for the delivery of genetic material into human cells and as such there is demand for high-titre manufacturing processes. The key to the successful development of such processes are analytical methods that can be applied to the final purified samples and throughout the production process. Many conventional methods for quantitative analysis of adenoviruses are labour and time-intensive. For example, a plaque assay can take up to 7 days to perform, is prone to error and will only report the number of infectious and not total viral particles. The resolving power of the high-performance liquid chromatography (HPLC), on the other hand, permits separation of intact virus particles from other cellular contaminants or virus particle fragments.
Anion-exchange chromatography has already been applied to analyse various adenovirus preparations. The results from the anion-exchange HPLC methods can be obtained much faster, within minutes, thus allowing for a faster evaluation of different process steps. A method was designed and developed to quantify adenoviral particles using a strong anion-exchange CIMac™ Analytical column. Regeneration conditions were incorporated to extend the functional life of the column.
Bacteriophages are used in a broad range of applications, including phage therapy and phage display. With the growing problem of antibiotic resistance leading to untreatable bacterial infections, they are becoming very interesting as antimicrobial agents, not only in medicine, but also in veterinary medicine, food industry and agriculture. Phages intended for use as antimicrobial agents, especially those for human use, need to be purified of contaminants.
Here we present efficient single step purification method for a Staphylococcus aureus phage VDX-10 from bacterial lysate on a CIM® QA Disk Monolithic Column (Figure 1). The described method can be used also on a larger scale using a CIM® QA-8 mL Tube Monolithic Column (Figure 2).
Filamentous phage M13 is a rod shaped non-lytic bacterial virus. M13 genetic material is used for many recombinant DNA processes, and the virus has also been studied for its uses in nanostructures and nanotechnology. The phage has been intensively studied for purposes of phage display and as a delivery vehicle for gene therapy. Phage display was first demonstrated with M13 bacteriophages and the filamentous phage remains a workhorse for this technology. Because of its typical size and rod shape it is considered as a challenging for purification. With large and highly interconnected pores monolithic chromatographic supports are also bridging that problem.
The ability to improve the purification process of M13 and other phages can have a significant impact on the market. By using phages for gene therapy, there will be a decrease in manufacturing time and production costs while enhancing the gene insertion. For phage display, a quicker method for phage purification will allow this powerful tool, which shortens the new drug discovery path and illuminates the basic interactions between different proteins, to be used with higher frequency.
Virus like particles (VLPs) are particles that structurally resemble viruses but do not contain any genetic material. They are formed when structural viral proteins spontaneously self-assemble in transfected cells. After VLPs are formed they need to be purified. Since the extract from expressing cells contains not only VLPs but also cellular DNA and proteins, VLPs purification represents a great challenge for the downstream processing.
Adenovirus serotype 3 dodecahedric virus-like particles (Ad3 VLPs) are an efficient vector for the delivery of the anticancer antibiotic drug bleomycin (BLM) – the use of Ad3 VLPs results in over 100 fold improvement of BLM bioavaliability. Ad3 VLPs are formed from penton bases of the adenovirus serotype 3 (Ad3) after these penton bases are expressed in a baculovirus/insect cell system. Ad3 VLPs are approximately 28 nm in size and have a molecular mass of 3.6 MDa. The current purification process of Ad3 VLPs consists of two purification steps, ultracentrifugation with a sucrose gradient (step 1) and ion-exchange chromatography (step 2) on Q-Sepharose and the whole procedure takes 5 days. Since Ad3 VLPs are large biomolecules, monolithic technology was applied for their purification with the aim to speed up the purification process.
Virus like particles (VLPs) are particles that structurally resemble viruses, but do not contain any genetic material. They are formed when structural viral proteins spontaneously self-assemble in transfected cells. Extracts from expressing cells contain not only VLPs, but also cellular DNA and proteins. These need to be removed in order to obtain pure VLPs, which are then applied for the production of vaccines, as delivery systems, as well as in other fields of nanotechnology applications (for the application on DSP of Ad3 VLPs check the Application Note A029). The purity of the final VLPs product is evaluated by methods like SDS-PAGE, agarose electrophoresis, PicoGreen analysis, BCA or Bradford assay.
In this work, CIMac™ QA Analytical Column was used for in-process control of the adenovirus serotype 3 dodecahedric virus-like particles (Ad3 VLPs). Samples obtained from different purification steps were injected on the CIMac™ QA Analytical Column and elution profiles were compared.
Environmental water is contaminated with human enteric viruses through the discharge of sewage contaminated water. As a consequence, they are present in various environmental water sources: irrigation water, wastewater, recreational water, ground or subsurface water, and even drinking water. The continuous low level transmission of these viruses can result in the spread of some viral infections. The nature of most enteric virus diseases is such that they elude epidemiological studies. Improved detection of viruses that are present in low concentration could prevent a considerable number of infections. Among the most important human food-borne viruses are Noroviruses (NoVs), members of Caliciviridae family and hepatitis A virus (HAV) which can be the source of serious outbreaks.
CIM® monolithic columns in combination with ultracentrifugation and RT-qPCR were used for the concentration and detection of hepatitis A virus and feline caliciviruse, a norovirus surrogate. At the same time efficiency of newly developed method was compared with reference method, based on membrane filter.
One of the most important plant viruses causing great economical losses in potato production is the filamentous Potato virus Y (PVY); virion size is 740 nm × 11 nm. Preparation of the pure virus suspension is essential for in vitro characterisation of the virus and also in many applications (e.g. antibody production). Virus purification usually consists of complicated and time-consuming protocols involving several ultracentrifugation steps, which are needed for isolation of the virus from the complex plant tissue matrix.
Different column chemistries, mobile phases and sample preparation strategies were examined during the method development study. Based on the obtained results, an optimised purification method for PVY from plant tissue on a CIM® QA Disk Monolithic Column was designed. The presence of the virus in the chromatographic fractions was monitored with viral RNA quantitation (RT-qPCR), viral protein detection (SDS-PAGE) and observation of the viral particle integrity (transmission electron microscopy).